Pemphigus is a life-threatening, antibody-mediated skin disease in which acantholysis (loss of epidermal cell-to-cell contacts) results in blister formation. Patients with pemphigus develop antibodies which bind the keratinocyte cell surface, the site of primary pathology. These antibodies, in the absence of complement, are capable of causing acantholysis and are probably pathogenic. The purpose of this project will be to 1) characterize the antigens with which different pemphigus sera react, 2) determine if antigenic specificity is correlated with different clinical types of pemphigus, and 3) ultimately, understand the molecular basis of the antigen-antibody reaction which triggers acantholysis at different levels in the epidermis (i.e. superficially in pemphigus foliaceus, deep in pemphigus vulgaris). Indirect immunofluorescence will be used to detect pemphigus antigens in cultured keratinocytes under varying conditions which modulate differentiation. Distinct patterns of fluorescence for different groups of sera would suggest that distinct antigens are being detected and would identify the optimal culture conditions to use to further characterize them. In order to demonstrate synthesis of these antigens and to characterize them, pemphigus antigens will be immunoprecipitated from cell cultures radiolabeled with metabolic precursors, and identified by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), and fluorography. In order to show that specifically identified molecules in culture are present in the normal human epidermis, detergent extracts of suction blister-dirived epidermis will be electrophoresed on SDS gels, transferred to nitrocvellulose paper, and specific pemphigus bands will be identified by immunoperoxidase staining. Further characterization and comparison of pemphigus antigens will be done by lectin binding profiles, one-dimensional peptide mapping by limited proteolysis, and reactivity with monoclonal antibodies. Functional studies will determine if there is antigenic specificity of antibodies which can cause acantholysis in organ culture. Ultimately we hope to understand the molecular basis of acantholysis in general and of different clinical types of pemphigus in particular.